The Protective Effect of Thai Fermented Pigmented Rice on Urethane Induced Somatic Mutation and Recombination in Drosophila Melanogaster

Full Length Research Paper

The protective effect of Thai fermented pigmented rice on urethane induced somatic mutation and recombination in Drosophila melanogaster

Yossaporn Plaitho, Kaew Kangsadalampai* and Monruedee Sukprasansap

Institute of Nutrition, Mahidol University, Nakhon Pathom 73170, Thailand.

Accepted 25 September, 2012

This study was aimed to proof that fermentation could increase phenolic and anthocyanin contents, antioxidant activities and antimutagenicity of Thai pigmented rice. Six cultivars of rice (Sung Yod, Mon Poo, Hom Mali Daeng, Hom Nil, Riceberry, and black glutinous rice) were fermented with a mixed culture of yeasts and molds (Look-Pang) at 30°C for 3 days to produce six types of fermented rice (Khao-Mak). Each sample exhibited significantly higher total phenolic and anthocyanin contents as well as antioxidant activities, including 2,2 ′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP), than that of the unfermented one of the same variety. Neither fermented nor unfermented pigmented rice substituted for corn flour in the fly medium was mutagenic in the somatic mutation and recombination test using Drosophila melanogaster . Interestingly, the antimutagenicity of fermented rice against urethane in the same testing was proven to be greater than that of its corresponding unfermented rice. This might be due to the higher content of total phenolic compounds, anthocyanins and antioxidant activities in fermented rice probably because of the catalytic action of enzymes produced by the starter organisms during fermentation which are capable of hydrolyzing glucosides of the inactive components to the active aglycones.

Key words: Fermented rice, total phenolic, anthocyanin, antioxidant activities, antimutagenicity.

INTRODUCTION

Rice is a staple food and an economic crop of Thailand. It Khao-Mak is a traditional fermented rice in Thailand is not only white rice but also many special rice cultivars that is made of white glutinous rice fermented with Look- that contain color pigments, such as black glutinous rice Pang at room temperature for 3 days (Lotong, 1992). (black rice), Hom Nil rice (purple rice) and Sung Yod rice Look-Pang is a microbial starter containing a mixed (red color). Vichapong et al. (2010) revealed that varieties culture of Aspergillus species, Rhizopus species and of Thai pigmented rice such as black glutinous rice and Mucor species, together with Saccharomyces cerevisiae Hom Mali Daeng had higher phenolic compounds, total and Candida species inoculums in rice flour mixed with flavonoid and antioxidant activity than normal white staple herbs such as pepper, garlic and galangal as rice. Anthocyanins are commonly a group of pigments antibacterial agents (Manosroi et al., 2011). Enzymes found in pigmented rice such as purple, black and red from the molds hydrolyze starch in the rice to sugars, rices. These compounds have many biological properties which are partially fermented to alcohol by the yeast. such as scavenging free radicals (Wang and Jiao, 2000), Organic acids (e.g. lactic acid) are also produced prevention of DNA damage in cancer cells (Hou, 2003), (Lotong, 1992). The product gives sweet taste, a little activating glutathione-related enzymes and alcohol and lactic acid flavor, soft texture, with lumps of NAD(P)H:quinone reductase (Shih et al., 2007; Singletary cooked glutinous rice and succulent grain. Black et al., 2007). glutinous rice is sometimes substituted for white glutinous rice to produce Khao-Mak since it is a rich source of phytochemicals such as anthocyanins (Sompong et al., 2011). Interestingly, fermented black glutinous rice *Corresponding author. E-mail: [email protected]. Tel: increased its antimutagenicity against in vivo formed +66 2 800 2380/399. Fax: +66 2 441 9344. nitrosomethylurea in somatic mutation and recombination 92 J. Med. Plants Res.

test (SMART) (Vipassanatham et al., 2012) and nitrite compounds) with dimethylsulphoxide (DMSO) (10 ml) for 30 min in treated 1-aminopyrene on Salmonella typhimurium TA98 a shaking water bath (30°C) according to the method of Omata et (Sadabpod et al., 2010) compared with that of its al. (2009). Then, the mixture was centrifuged at 2500 ×g for 10 min and the supernatant was collected. The residue was re-extracted corresponding raw and cooked rice. under the same condition. The combined supernatant was In this investigation urethane or ethyl carbamate was determined for its antioxidant activities using the DPPH assay as used as a positive mutagen in the somatic mutation and described by Fukumoto and Mazza (2000), and the ferric reducing recombination test as suggested by Abraham and Graf antioxidant power (FRAP) assay according to the procedure (1996). It is a natural constituent of tobacco leaves and described by Griffin and Bhagooli (2004). The total phenolic content of the extract was determined according to the method described tobacco smoke (Schmeltz et al., 1978) and is also by Amarowicz et al. (2004). The anthocyanin content of each present in fermented food products such as bread, yogurt sample extracted with acidified ethanol was determined according and cheese (Ough, 1976; Canas et al., 1989; Miller and to the method described by Abdel-Aal and Hucl (1999). Miller, 1983). In rodents, urethane was found to produce lymphomas, lung tumors, hepatomas and melanomas (Mori et al., 2000; Mirvish, 1968; International Agency for SMART

Research on Cancer (IARC), 1974) and was also found Two strains of Drosophila melanogaster were used. Males of to induce point mutation, gene conversion, mwh/mwh and females of ORR;flr 3/In(3LR) TM3, ri p p sep l(3)89Aa intrachromosomal recombination, chromosomal bx 34e e Bd S, Ser were obtained from the Institute of Toxicology, aberration and sister chromatid exchanges in yeast, plant Swiss Federal Institute of Technology and University of Zurich. The system and mammalian cells (Schlatter and Lutz, 1990; test was performed as described by Graf et al. (1984). One hundred 3 days old trans-heterozygous larvae ( mwh flr +/mwh TM3 ) were Uggla and Busk, 1992). IARC classified urethane as transferred to the Drosophila medium (Roberts, 1986) in which each possibly carcinogenic to humans (group 2B) (IARC, sample was substituted for corn flour (used for the mutagenicity 1974). Therefore, the objective of this study was to evaluation) or the Drosophila medium in which each sample was determine the total phenolic content, anthocyanin content substituted for corn flour and 20 mM urethane was substituted for and antioxidant activities of fermented pigmented rice as water (used for antimutagenicity evaluation). The standard medium well as the possible antimutagenicity of different types of was used as the negative control while the medium containing 20 mM urethane was used as the positive control. Each experiment fermented pigmented rice in the somatic mutation and was done twice. recombination test. The antimutagenicity of each sample was determined from the percentage of inhibition calculated as follows: percentage of inhibition = ((a-b)/a)x100, where “a” is the frequency of spots MATERIALS AND METHODS induced by urethane alone and b is the frequency of spots induced

Chemicals by urethane in the presence of the sample. It is proposed that percentages of inhibitions less than 20, 20-40, 40-60 and more than Urethane, 2, 4, 6-tripyridyl-s-triazine (TPTZ), ferric chloride 60 are the evidences of negligible, weak, moderate and strong hexahydrate and ferrous sulfate heptahydrate were purchased from antimutagenicity, respectively as suggested by Kruawan et al. Sigma Chemical (St. Louis, MO, USA). Diethylether and sodium (2012). acetate trihydrate were purchased from Merck (Darmstadt, Germany). Propionic acid, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid and Folin-Ciocalteu reagent were supplied from Fluka Statistical analysis Chemika (Buchs, Switzerland). Trolox was bought from Aldrich Chemical (Milwaukee, WI, USA). All other chemicals and reagents Total phenolics, anthocyanins and antioxidant activities data were were of analytical grade. reported as means + standard deviations for triplicate analyses of the same sample. One-way analysis of variance (ANOVA) and Schefee’s multiple comparison tests were carried out to test for Preparation of fermented pigmented rice differences among the means of fermented rice using Statistical Package for Social Sciences (SPSS, version 13). The mean values Six cultivars of pigmented unpolished rice, namely, Sung Yod (red of unfermented rice and those of fermented rice were analyzed by rice), Mon Poo (red rice), Hom Mali Daeng (red rice), Hom Nil paired-samples t-test. Statistical significance was considered for p (purple rice), Riceberry (purple rice) and black glutinous rice (black less than 0.05. For SMART, the wings of the surviving flies were rice) were obtained from local markets in Bangkok. A commercial analyzed for the occurrence of mutant spots and evaluated as starter called Look-Pang was bought from ‘100 Year Samchuck described by Frei and Würgler (1988). The estimation of spot Market’ in Suphanburi province. Rice was fermented in Thai frequencies and confidence limits of the estimated mutation traditional style as suggested by Lotong (1992) with slight frequency were performed with significance level of α = β = 0.05. modification as the following. It was mixed with distilled water (1:2 w/v) and cooked in an electric cooker. Cooked rice was cooled at room temperature and fermented with Look-Pang (0.2 g/100 g of raw rice) at 30°C for 3 days in a glass container. The fermented rice RESULTS AND DISCUSSION was dried at 60°C in a hot air oven for 24 h and was stored under vacuum in a polyethylene bag at 4°C before use. Total phenolic and anthocyanin contents

The total phenolic content of fermented rice was higher Antioxidant activities, total phenolic and anthocyanin contents than that of its corresponding unfermented one (Figure of fermented pigmented rice 1A). Unfermented rice had an average of 431.6 to 1575.6 Ground sample (1 g) was extracted (both hydrophilic and lipophilic mg gallic acid equivalent/100 g dry weight, whereas Plaitho et al. 93

Figure 1. Total phenolic content (A) and anthocyanin content (B) of unfermented and fermented pigmented rice. Values are presented as means ± SD (n = 3). Data with different upper case letters indicate significant differences (p < 0.05) between unfermented and fermented rice of the same varieties, while those with different lower case letters indicate significant differences (p < 0.05) between fermented rice varieties.

fermented rice had an average of 1026.4 to 2086.9 mg anthocyanin content of unfermented rice samples of this gallic acid equivalent/100 g dry weight. The highest total study ranged from 0.4 to 111.1 mg cyanidin3- phenolic content (2086.9 mg gallic acid equivalent/100 g glucoside/100 g dry weight, whereas those of the dry weight) belonged to fermented Riceberry. The fermented rice samples ranged from 1.5 to 122.2 mg 94 J. Med. Plants Res.

cyanidin 3-glucoside/100 g dry weight (Figure 1B). antioxidant activity (5.3 mmol Trolox equivalent/100 g dry Fermented black glutinous rice exhibited the highest weight) was found in fermented black glutinous rice. The anthocyanin content with an approximately 122.2 mg FRAP values of unfermented rice ranged from 0.9 to 1.9 cyanidin 3-glucoside /100 g dry weight in this study. mmol Fe(II)/100 g dry weight, whereas those of The increases of total phenolic and anthocyanin fermented rice ranged from 1.3 to 2.9 mmol Fe(II)/100 g contents of fermented samples in this study were similar dry weight (Figure 2B). It was noted that fermented rice to those found by other investigators who worked on had significantly higher reducing abilities than that of some other plants. Amongst those, Kwak et al. (2007) unfermented rice of the same variety. The fermented found that Chungkookjang, a fermented soybean paste in black glutinous rice had the highest FRAP values (2.9 Korea, had significantly higher total polyphenol content mmol Fe(II)/100 g dry weight). than that of raw soy bean. Lee et al. (2007, 2008) Our finding on the benefit of fermentation was reported that fermented black beans exhibited higher consistent with the observations of other researchers. total phenolic and anthocyanin contents than those of Lee et al. (2008) indicated that black bean fermented with unfermented black beans. Furthermore, Liang et al. Aspergillus spp. exhibited higher levels of DPPH (2009) found that Phellinus -fermented rice exhibited scavenging activity and Fe 3+ reducing power than the higher total phenolic content than that of unfermented non-fermented one. Moktan et al. (2008) found that rice. Sadabpod et al. (2010) also reported that total soybean fermented with Bacillus subtilis was more phenolic contents of fermented Hom Nil rice and black effective in DPPH scavenging activity and Fe 3+ reducing glutinous rice were higher than those of both raw rice and ability than that of unfermented one. Moreover, cooked rice. Sadabpod et al. (2010) reported that DPPH scavenging The greater total phenolic and anthocyanin contents of activity and Fe3+ reducing power of fermented Hom Nil fermented rice may be due to the enzymatic activities of rice and black glutinous rice were higher than those of starter organisms in Look-Pang such as S. cerevisiae , both raw rice and cooked rice while fermented maize Aspergillus spp. and Rhizopus spp. It was reported, that (Daker et al., 2008) and Phellinus -fermented adlay and rubi fructus fruit (Ju et al., 2009), buckwheat, wheat germ, rice (Liang et al., 2009) had better DPPH scavenging barley, rye (Ðordevi ć et al., 2010) and watermelon rind effect than that of unfermented ones. S. cerevisiae (Erukainure et al., 2011) fermented with S. cerevisiae and fermented watermelon rind was shown to have higher koji made from black soybean fermented with Aspergillus DPPH scavenging activity and Fe 3+ reducing power than sp. or Rhizopus sp (Lee et al ., 2008) exhibited higher that of unfermented one (Erukainure et al., 2011). total phenolic content than those of unfermented ones. It Enhancing the DPPH scavenging activity and Fe 3+ was suggested that the increase of total phenolic content reducing power of fermented rice might be due to the might be due to hydrolysis by a glycoside hydrolase increase of total phenolic and anthocyanin contents (as during fermentation (Ju et al., 2009). Lee and Chou discussed earlier). Anthocyanins are natural phenolic (2006) revealed that fermentation caused a marked pigments that were reported to scavenge free radicals - increase in the content of aglycone (daidzein, glycitein, such as superoxide (O 2 ), singlet oxygen (˙O 2), peroxide - and genistein), the bioactive isoflavone, and a significant (ROO ), hydrogen peroxide (H 2O2) and hydroxyl radical reduction in the content of beta -glucoside isoflavone (OH -) (Wang and Jiao, 2000). Normally, the antioxidant (daidzin, glycitin, and genistin), compared with the activity of the anthocyanidins (aglycons) was generally unfermented steamed black bean. The increase of greater than those of the corresponding anthocyanins aglycone content and the increase of beta-glucosidase (glycosides) (Wang and Stoner, 2008). It should be noted activity during the fermentation of this koji showed a that other antioxidants commonly found in pigmented rice similar trend. Therefore, the action of enzyme such as such as ferulic acid, p-coumaric acid, protocatechuic beta-glucosidase produced by the starter organism acid, vanillic acid, caffeic acid (Vichapong et al., 2010; during fermentation might be an important factor Sompong et al., 2011) and tocopherols may exhibit contributing to the increase of phenolic and anthocyanin antioxidant activity (Aguilar-Garcia et al., 2007; Hyogo et contents of fermented rice. al., 2010; Vichapong et al., 2010; Sompong et al., 2011).

Antioxidant activities of unfermented and fermented Mutagenicity and antimutagenicity of fermented rice rice Substituting each sample for corn flour (50, 75 and The DPPH antioxidant activity expressed as mmol Trolox 100%) in the Drosophila medium for rearing the trans- equivalent/100 g dry weight of each sample as shown in heterozygous larvae gave adult flies with normal wing Figure 2A. Unfermented rice had an average of 2.3 to 4.3 hairs indicating that none of the samples contained mmol Trolox equivalent/100 g dry weight while fermented mutagenic compound (data not shown). Interestingly, the rice had an average of 3.4 to 5.3 mmol Trolox counteracting effect against urethane mutagenicity of equivalent/100 g dry weight. The highest DPPH unfermented rice and fermented rice on D. melanogaster Plaitho et al. 95

BBB

Figure 2. DPPH antioxidant activity (A) and ferric reducing antioxidant power (B) of the unfermented and fermented pigmented rice. Values are presented as means ± SD (n = 3). Data with different upper case letters indicate significant differences (p < 0.05) between unfermented and fermented rice of the same varieties, while those with different lower case letters indicate significant differences (p < 0.05) between fermented rice varieties.

was obtained (Figure 3). It has shown that fermented rice rice of the same variety. Sung Yod, Mon Poo, Hom Mali gave greater inhibitory effect than that of unfermented Daeng and Hom Nil varieties exhibited negligible 96 J. Med. Plants Res.

Figure 3. Antimutagenicity of unfermented and fermented pigmented rice against urethane in the somatic mutation and recombination test. It is proposed that percentage of inhibition less than 20, 20–40, 40–60 and higher than 60 indicates negligible, weak, moderate and strong antimutagenicity, respectively (Kruawan et al, 2012).

antimutagenicity (< 20% inhibition), while Riceberry and increase in phenolic contents such as anthocyanins black glutinous rice had weak antimutagenicity (20 to during fermentation might contribute to the increase of 40% inhibition) in both trials 1 and 2. The antimutagenicity of fermented rice. Anthocyanins were antimutagenicity effect of fermented rice increased as found to protect N-nitrosomethylbenzylamine (Reen et Mon Poo, Hom Nil and Hom Mali Daeng turned to be al., 2006), tert-butyl hydroperoxide (t-BHP) (Hwang et al., weakly antimutagenic, while Sung Yod, Riceberry and 2011a) and dimethylnitrosamine (Hwang et al., 2011b) in black glutinous rice turned to be moderately inducing hepatic damage in rats. In addition, Choi et al. antimutagenic (40 to 60% inhibition). (2009) showed that anthocyanin obtained from purple- Increasing antimutagenicity of pigmented rice via fleshed sweet potato protected against acetaminophen- fermentation, as observed in this study, is compatible induced hepatotoxicity by blocking CYP2E1-mediated with other studies. For example, Sadabpod et al. (2010) acetaminophen bioactivation. It is suggested that the revealed that fermented black glutinous rice had higher protective effects of fermented rice against urethane- antimutagenicity against nitrite treated 1-aminopyrene on induced mutagenicity may be due to the ability of S. typhimurium TA98 than that of its corresponding raw anthocyanins to inhibit CYP2E1-mediated urethane and cooked rice. Vipassanatham et al. (2012) also metabolism since Hoffler et al. (2005) reported that indicated that black glutinous rice, after being fermented, urethane-induced micronuclei formation was reduced in had higher antimutagenicity against in vivo formed CYP2E1-null mice. However, other compounds in nitrosomethylurea in D. melanogaster as compared to pigmented rice such as ferulic acid, p-coumaric acid, that of raw and cooked rice. In addition, many protocatechuic acid, vanillic acid, caffeic acid (Hyogo et researchers also found the benefit of fermentation on al., 2010; Vichapong et al., 2010; Sompong et al., 2011) other foods such as fermented soymilk (Hsieh and Chou, might also contribute to the enhancing antimutagenicity. 2006), soybean koji (Lin and Chou, 2006) and fermented This study revealed that fermentation influenced the black bean (Hung et al., 2007) that could suppress the observed antimutagenicity against urethane and the mutagenicity of 4-nitroquinoline-N-oxide. levels of antimutagenicity depended on rice variety. The Our investigation also suggested that a remarkable high bioactivation (HB) cross of D. melanogaster used in Plaitho et al. 97

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